STUDIES ON THE CYCLIC AMP RECEPTOR PROTEIN OF E COLI

cAMP 受体蛋白 (CRP) 在分解代谢物的转录中发挥关键作用 通过 cAMP 依赖性 DNA 作为正向调节因子的可抑制操纵子 （启动子）结合。 拟议的研究涉及对角色的调查 CRP 结构和 cAMP 与 DNA 结合的功能。 CRP有一个域 其中氨基近端区域参与结合 cAMP 的结构 羧基近端区域参与亚基-亚基相互作用 DNA 结合。 多肽片段和核心是通过有限的方法产生的 CRP的蛋白水解；这些将在 CRP 重建方面进行研究 体外 lac 和 DNA 的活性、DNA 结合和可能的调节特性 gal操纵子表达。 CRP的羧基末端修饰 将进行羧肽酶以确定该区域在DNA中的作用 结合和CRP构象。 将继续进行化学改性研究 确定对 CRP 结构和功能的影响。 温度敏感的 CRP 突变体将被纯化并根据 ts 损伤进行表征；回复体 将选择表现出与野生动物不同的特性进行研究 键入 CRP。 使用杂交瘤将制备针对CRP的单克隆抗体 碎片。 这些单特异性抗体将用作 CRP 探针 构象和功能。

The cAMP receptor protein (CRP) plays a key role in transcription of catabolite repressible operons as a positive regulatory factor via cAMP dependent DNA (promoter) binding. The proposed research involves an investigation of the role of CRP structure and function for cAMP and DNA binding. CRP has a domain structure in which the amino proximal region is involved in binding cAMP and subunit-subunit interaction while the carboxyl proximal region is involved in DNA binding. Polypeptide fragments and cores have been generated by limited proteolysis of CRP; these will be studied with regard to reconstitution of CRP activity, DNA binding and possible regulatory properties for in vitro lac and gal operon expression. Modification of CRP at the carboxyl terminus by carboxypeptidase will be carried out to determine the role of this region in DNA binding and CRP conformation. Chemical modification studies will be continued to determine effects on CRP structure and function. A temperature sensitive CRP mutant will be purified and characterized in terms of the ts lesion; revertants will be selected for study which show properties different from that of wild type CRP. Using hybridomas monoclonal antibodies will be prepared against CRP fragments. These monospecific antibodies will be used as probes of CRP conformation and function.